Technologies for amplifying or detecting nucleic acids have been indispensable in the fields of medicine; pharmacy; agriculture, forestry, and fishery; and food, for example, as experimental tools not only in genetic engineering and molecular biology, but also in the diagnosis of diseases, examination of genetically modified foods, and detection of microorganisms, viruses, and pathogenic bacteria.
It is known that, regardless of their biological species, there is a group of genes which share high homology in genes that encode substances, enzymes, and others having similar functions. It is also known that the individual genomes of organisms belonging to the same species are not perfectly identical and have differences in their nucleotide sequences which are referred to as polymorphism. The polymorphism is known to exhibit a deletion or an insertion of one to several ten nucleotides, a duplication of a specific nucleotide sequence, and the like. Substitutions of a nucleotide with another nucleotide are referred to as a single nucleotide polymorphism (SNP; which may be described herein as a single nucleotide substitution) and have attracted attention as an indicator for searching genes related to diseases, determining the susceptibility to diseases, or understanding differences in the sensitivity to drugs (effects and side effects). Studies are also underway on methods for the detection of such substitutions.
Methods for detecting a particular gene from a group of genes sharing high homology and for detecting an SNP include a method in which an RNA-DNA hybrid formed by hybridization of an RNA-containing probe and a target. DNA is subjected to cleavage with ribonuclease (for example, Patent Document-1). In this method, the RNA-containing probe is designed such that the hybrid is not cleaved with ribonuclease when there is a mismatch between the probe and a target DNA, thereby allowing one to ascertain the presence or absence of a specified nucleotide or a nucleotide substitution in the target DNA.
Conventional probes for gene detection have been designed by selecting such a nucleotide sequence that specific hybridization occurs between the probe and a target nucleic acid, using as an indicator the Tm value of a hybrid which is to be formed or the self-complementarity of the probe, based on the nucleotide sequence of the target nucleic acid. A variety of software for designing appropriate probes is also provided.
However, the RNA-containing probe as described above which is for detecting a target nucleic acid can be designed only in a region which includes or does not include a specified nucleotide, or which includes a specified nucleotide at which a nucleotide substitution is caused or is likely to be caused, and thus has a limitation in selecting regions, as compared with cases where conventional probes are designed. Further, it is required that the sensitivity of the RNA portion contained in the probe to ribonuclease H varies to a great extent, depending upon the presence/absence of a specified nucleotide or SNP. Up until now, these limitations have forced researchers to have no choice but to design RNA-containing probes on the basis of their experiences or under trial and error.